Title: Damage sensor role of UV-DDB during base excision repair. PI: Bennett Van Houten, PhD Abstract/Summary UV-damaged DNA-binding protein complex (UV-DDB) is a damage sensor for the detection of UV- induced photoproducts in chromatin and consists as a heterodimer of DDB2, a 48 kDa DNA damage recognition protein, and DDB1 a 127 kDa protein that together with Cul4A and RBX1 forms an E3 ligase. UV-DDB was believed to be solely involved in the initial steps of global nucleotide excision repair (NER) of environmentally-induced photoproducts in the context of chromatin by ubiquitylating histone 2A to allow relaxation of the nucleosome around the lesion and subsequent access by NER proteins such as XPC and TFIIH. This highly innovative project consisting of three aims and seeks to examine the role of human UV-DDB in recognizing oxidative base damage or base excision repair (BER) intermediates in naked DNA or in the context of nucleosomes. We hypothesize that UV-DDB is a general damage sensor and can stimulate APE1 and OGG1 activities on abasic sites and 8-oxo-dG adducts, respectively. We also hypothesize that PARP1 PARylation of UV-DDB will alter these interactions. Aim1 uses biochemical approaches to study how UV-DDB stimulates these enzymes to incise lesions embedded in naked DNA or a nucleosome and will also study the effects of PARylation. The second aim, uses single molecule analysis to follow UV-DDB recognition and co-localization with either APE1 or OGG1 on DNA damage arrays using fluorescence microscopy and Qdot labeled proteins. The third aim uses a highly innovative approach to introduce 8-oxo-dG at specific regions in the genome to follow UV-DDB recruitment and co-localization with OGG1 and APE1. Finally using Bio- ID approaches we will explore the UV-DDB interactome during BER. This project will give an unprecedented view of the complex process of damage recognition steps of human base excision repair and answer several key questions regarding damage recognition that have been intractable in the absence of single molecule approaches. Completion of this project will have a long and lasting impact on the field.